Enterococci have become important nosocomial pathogens over the past 25 years, with Enterococcus faecium infections recently increasing from less than ten percent of clinical isolates to causing over one-third of all enterococcal infections. Several surface proteins with a cell wall anchoring LPxTG motif are associated with enterococcal infection. Recently, researchers have indicated the probability of a new cell wall binding motif, designated the WxL domain. Proteins containing this domain have been implicated as important in peritonitis infections caused by Enterococcus faecalis and aggregation in Lactobacillus coryniformis. My preliminary data have shown that the WxL proteins of E. faecium exist in putative operons containing a transmembrane protein and an LPxTG protein, indicating the possibility of a novel form of surface assembly. The long term goal of this proposal is to better understand cell wall associated proteins and their possible contribution to colonization and virulence in Enterococcus faecium. The goals of this study concentrate on proteins containing the newly identified WxL domain at the C-terminal region. The specific aims of the proposed study will first investigate the role of WxL proteins in E. faecium pathogenesis by looking at mutants in a number of in vitro models of virulence related functions and in vivo models of typical infections caused by Enterococcus faecium. Second, the function of the WxL domain will be defined, the localization of the proteins examined, and an initial understanding of the interactions between the proteins within the putative operons will be gained. Lastly, the in vivo expression of these proteins will be analyzed, their antigenicity determined, and the possibility of protecting against E. faecium infection using WxL proteins will be explored. This proposal encompasses the microbiology and infectious disease fields, specifically concentrating on the emerging pathogen E. faecium. This study will contribute to the knowledge of this normally commensal organism, as well as explore future options for immunization. The proposed study includes a variety of techniques used in numerous fields including molecular biology, genetics, biochemistry, immunology, and structural biology and should cover the duration of the last three years of Ph.D. training. PUBLIC HEALTH RELEVANCE: Although Enterococcus faecium is an important hospital-associated pathogen, research on how this normally commensal organism initiates disease and interacts with the host is lacking when compared to the information known about other gram positive pathogens. Assessment of the importance of putative surface proteins with the "WxL" motif in virulence, identification of the binding domains involved in cell wall association, possible protein-protein interactions between proteins encoded in the same operon, expression of the genes encoding WxL proteins within the host, and the possibility of using the WxL proteins in immunization for E. faecium infections will enhance the very limited knowledge of the mechanisms for pathogenicity of this organism, and may provide for possible immunization candidates for the prevention of enterococcal infections.